|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Cytoskeletal rearrangements in human red blood cells induced by snake venoms: light microscopy of shapes and NMR studies of membrane function
Tsz Wai Yau*†, Rhiannon P. Kuchel‡, Jennifer M. S. Koh*, David Szekely§, Peter J. Mirtschin¶ and Philip W. Kuchel1*‖
*School of Molecular Bioscience, University of Sydney, Sydney, NSW 2006, Australia, †Australian Nuclear Science and Technology Organisation, Lucas Heights, Sydney, NSW 2232, Australia, ‡Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia, §Mark Cowley Lidwill Program in Cardiac Electrophysiology, The Victor Chang Cardiac Research Institute, Sydney, NSW 2010, Australia, ¶School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5001, Australia, and ‖Singapore Bioimaging Consortium, BioMedical Sciences Institutes, Biopolis, Singapore 138667, Singapore
RBCs (red blood cells) circulating through narrow blood capillaries withstand major deformation. The mechanical and chemical stresses commonly exerted on RBCs continue to attract interest for the study of membrane structure and function. Snake venoms are lethal biochemical ‘cocktails’ that often contain haemotoxins, metalloproteinases, myotoxins, neurotoxins, phosphodiesterases, phospholipases and proteases. We have monitored the effects of 4 snake venoms (Pseudechis guttatus, Oxyuranus scutellatus, Notechis scutatus and Naja kaouthia) on human RBCs using NMR spectroscopy, DIC (differential interference contrast) and confocal light microscopy. RBCs underwent reproducible stomatocytosis, with unusual geographical-like indentations, spherocytosis, followed by rapid lysis. Confocal micrographs using a fluorescent dye linked to phalloidin showed that the change in morphology was associated with the aggregation of actin in the cytoskeleton. 31P NMR saturation transfer experiments recorded transport of the univalent anion HPA (hypophosphite) on a subsecond time scale, thereby reporting on the function of capnophorin or Band 3 linked to the cytoskeleton; anion-exchange activity was substantially reduced by venom treatment. We propose a molecular-cytological hypothesis for the shape and functional changes that is different from, or supplementary to, the more ‘traditional’ bilayer-couple hypothesis more often used to account for similar morphological changes invoked by other reagents.
Key words: bilayer-couple hypothesis, haemolysis, human red blood cell, light microscopy, phospholipase A2, red blood cell cytoskeleton, snake venom
Abbreviations: DIC, differential interference contrast, DMMP, dimethyl methylphosphonate, DNDS, 4,4′-dinitrostilbene-2,2′-disulfonate, FID, free-induction decay, Hb, haemoglobin, Hct, haematocrit, HPA, hypophosphite, PLA2, phospholipase A2, RBC, red blood cell, RF, radio-frequency, RP-HPLC, reverse-phase HPLC, TEP, triethyl phosphate, TFA, trifluoroacetic acid
1To whom correspondence should be addressed (email email@example.com).
Received 8 January 2011/7 July 2011; accepted 21 September 2011
Published as Cell Biology International Immediate Publication 21 September 2011, doi:10.1042/CBI20110012
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